Antibodies against Stachybotrys chartarum and methods for their use

ABSTRACT

Antibodies to various fungal antigens are disclosed, including monoclonal antibody 9B4 that selectively binds an antigen of  Stachybotrys chartarum  spores not found in  Stachybotrys chartarum  mycelium. The antibodies may be used in a variety of methods, such as detecting the presence of fungal antigens in the environment or within a sample obtained from an animal or plant, or testing the effectiveness of an agent in binding an antigen.

This is a national stage under 35 U.S.C. §371 of InternationalApplication No. PCT/US02/25493, filed Aug. 9, 2002, and claims thebenefit of U.S. Provisional Patent Application No. 60/311,458, filedAug. 10, 2001.

FIELD

This invention relates to binding agents, such as antibodies, that bindto target molecules, including cellular components of fungi.

BACKGROUND

Certain fungi found in indoor environments, including homes andbusinesses, may cause adverse health effects in people and animals byprovoking allergic reactions, causing infection, or releasing toxicsubstances. Recently, some attention has focused on the fungusStachybotrys chartarum, which grows in moist indoor environments.Exposure to this fungus has been implicated in respiratory,dermatological, gastrointestinal, and central nervous system disordersin humans and a variety of animals. See, e.g., Fung, F., et al.,Clinical Toxicology 36:79-86 (1998); Robbins, C. A., et al., AppliedOccupational and Environmental Hygiene 15:773-84 (2000). For example, ithas been suggested that Stachybotrys chartarum may be associated with“sick building syndrome,” an occupational condition in which workers aresickened by environmental toxins or pathogens. Craner, J., in:Bioaerosols, Fungi and Mycotoxins: Health Effects, Assessment,Prevention and Control, Johanning, E. (Ed.), (Eastern New YorkOccupational and Environmental Health Center, Albany, N.Y., 1999), pp.146-157.

Exposure to the toxic substances, such as trichothecenes, or allergensproduced by Stachybotrys chartarum is implicated in some respiratorydisorders, including inflammation and hemorrhage of the lungs, and alsomay increase disease susceptibility in exposed subjects by affecting theinflammatory and immune responses after exposure. See, e.g., Dearborn,D. G., et al., Environmental Health Perspectives 107:495-99 (1999);Flappan, S. M., et al., Environmental Health Perspectives 107:927-930(1999); Elidemir, O., Pediatrics 104:964-66 (1999); and Pitt, J. I.,British Medical Bulletin 56: 184-92 (2000). However, the exact role ofStachybotrys chartarum in causing these adverse health effects is notwell known and has been controversial, especially regarding humansusceptibility to Stachybotrys chartarum.

Accurate and precise monitoring methods are needed to better understandand analyze the role of Stachybotrys chartarum in certain diseases orconditions and the relevance of this fungus in causing or contributingto such health effects. Current methods of detecting this fungus rely onspore counts and cultivation of samples, which can be time-consuming andlabor-intensive, or use chromatographic detection of specificmycotoxins, which can be insensitive and often requires specializedequipment. Therefore, a need exists for a method of assessing exposureto Stachybotrys chartarum that is rapid, accurate, and efficient.

If available, an antibody that binds to an antigen on a fungus may beused to detect some fungi. An antibody-antigen complex, formed when anantibody binds to an antigen, may be detected using a variety oftechniques, such as the enzyme linked immunosorbent assay (ELISA),particle immunostaining, or fluorescence-based image analysis. Forexample, monoclonal antibodies against fungal allergens expressed byAlternaria alternata and several Aspergillus and Penicillium specieshave been used to identify and characterize environmental contamination.Chapman, M. D., et al., Clinical Reviews in Allergy and Immunology18:285-300 (2000). The application of these monoclonal antibodies formeasuring exposure was compromised by their cross-reactivity with otherfungal species, variability or lack of essential expression of allergensrecognized by the antibodies, or the lack of assay sensitivity due tolow level production of allergens per unit biomass. Mitakakis, T. Z., etal., Journal of Allergy and Clinical Immunology 107:388-90 (2001);Vailes, L., et al., Journal of Allergy and Clinical Immunology107:641-46 (2001). However, antibodies to Stachybotrys species, such asStachybotrys chartarum, and associated methods of detecting these fungiwere not previously known.

SUMMARY

Disclosed are antibodies to antigens produced by fungi commonly found inindoor environments (see Table 1), such as antibodies to an antigen ofStachybotrys chartarum, including antigens secreted by Stachybotryschartarum. Particular examples of such antibodies include monoclonalantibody (Mab) 9B4, which binds an antigen of Stachybotrys chartarumspores, or other antibodies that recognize an antigen to which 9B4specifically binds. Mab 9B4 does not react with spores or mycelium ofother fungi commonly found in indoor environments, including multiplespecies and/or isolates of Alternaria, Aspergillus, Aureobasidium,Botrytis, Cladosporium, Chaetomium, Curvularia, Fusarium, Memnoniella,Mucor, Myrothecium, Paecilomyces, Penicillium, Rhizopus, Scopulariopsis,Trichoderma, Ulocladium and Wallemia fungal genera, including thoseidentified in FIGS. 1-6. Mab 9B4 also does not react with spores ofother Stachybotrys species such as S. albipes, S. bisbyi, S.cylindiospora, S. echinata, S. kanipalenisis, S. microspora, S.nephrospora or S. subsimplex. Hence, Mab 9B4 can be consideredspecies-specific for spores of Stachybotrys chartarum. Other particularexamples of antibodies include Mabs 1D4, 3B2, 4E12, 9F9 and 10A5, whichreact with spores and/or mycelium of multiple fungal species, includingStachybotrys chartarum.

Monoclonal antibody 9B4 provides a specific test for the presence of, orexposure to, Stachybotrys chartarum spores. Cell lines producing theseantibodies, such as a hybridoma that produces the 9B4 monoclonalantibody, and labeled antibodies, such as antibodies carrying aradiolabel, biotin, or a fluorescent, calorimetric, or luminescentlabel, also are disclosed.

The antibodies may be used to identify and detect the presence of afungus within a sample obtained from the environment (e.g., soil, water,dust, bulk material, and air samples), a plant, or an animal.Additionally, since antigen may remain on or within an environmentallocus, animal, or plant after being contacted by a fungus, exposure tothe fungus may be detected even if the fungus later disintegrates ordisappears. The antibodies may be used to detect viable or dead fungi,fungal components, or soluble components, such as actively secreted orpassively released fungal products or immune complexes. Such fragmentsor components may be detected even after the physical or chemicalbreakdown of a fungus.

To detect exposure to the fungus, an interaction between the antigen andantibody is detected. In some instances, the sample is contacted with anantibody, and detection of an antibody-antigen complex indicates thepresence of antigen in the sample, either an antigen presentlyassociated with a fungus or an antigen that remained in the sample afterthe fungus contacted the sample. In other instances, the antibody isused to immunoprecipitate the corresponding antigen, and the isolatedantigen is used to screen a sample for the presence of other antibodiesto the antigen. For example, monoclonal antibody 9B4 may be used toisolate its cognate antigen, and that antigen may be used to detectexposure to Stachybotrys chartarum spores in animals by screening ablood sample for reactive antibodies.

The antibody also may be part of a kit for detecting a fungus or otherantibodies, such as a kit containing monoclonal antibody 9B4 fordetecting Stachybotrys chartarum spores. For example, Mab 9B4 may bepart of a kit to detect spores of S. chartarum in environmental samplesor to detect antibodies of similar specificity in animal samples.

The antibodies also may be used to test the effectiveness of an agent inbinding an antigen. In such embodiments, an antibody is mixed with anantigen and the agent, and antibody-antigen binding in the presence ofthe agent is compared to some reference standard or control. Forexample, monoclonal antibody 9B4 may be mixed with Stachybotryschartarum spores and an agent; if 9B4-spore binding in the presence ofthe agent is less than in a negative control (i.e., binding in theabsence of the agent), then the agent interferes with the 9B4-sporebinding and may effectively bind the same antigen as 9B4.

The detection of an immune complex formed between a Mab and itscorresponding fungal antigen, or the inhibition of the binding of a Mabto its antigen by competitive or blocking antibodies or antigens, may beaccomplished by several techniques. Such analytical techniques include,but are not limited to, assays using labeled or unlabeled reagents orantibodies. If labeled reagents and/or antibodies are used, the labelmay be a radioisotope, enzyme, metal, or a fluorescent, phosphorescentor chemiluminescent compound. Such assays include, for example,competitive and non-competitive homogenous and heterogenousenzyme-linked immunosorbent assays (ELISA) as symmetrical orasymmetrical direct or indirect detection formats; enzyme-linkedimmunospot assays (ELISPOT); radioallergosorbent tests (RAST);fluorescent tests, such as used in fluorescent microscopy and flowcytometry; Western, grid, dot or tissue blots; dip-stick assays; halogenassays; or protein arrays including antibody arrays. Other analyticaltechniques include immune complex-based assays such as immunodiffusion,precipitation, coagulation, agglutination and hemagglutinationtechniques as well as tests based on turbidimetry or nephelometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the cross-reactivity of severalmonoclonal antibodies (Mabs) with spores of different Stachybotrysspecies and different Stachybotrys chartarum isolates.

FIG. 2 is a graph illustrating monoclonal antibody (Mab) reactivity tomycelium of different Stachybotrys chartarum isolates.

FIG. 3 is a graph illustrating the cross-reactivity of several differentMabs against spores of fungal species commonly found in indoorenvironments.

FIG. 4 is another graph illustrating the cross-reactivity of severaldifferent Mabs against mycelium of several fungal species commonly foundin indoor environments.

FIG. 5 is a graph illustrating the sensitivity of Mab 9B4 againstdifferent Stachybotrys chartarum spores using enzyme-linkedimmunosorbent assay (ELISA).

FIG. 6 is a graph illustrating the effect on Mab binding after boilingand periodate treatment of the antigen.

The following abbreviations for fungal species are used herein; thenumbers following each species name indicate the spore concentrationsper well used in ELISA: Aal—Alternaria alternata, 6,750; Aca—Aspergilluscandidus, 115,000; Acl—Aspergillus clavatus, 875,000; Afl—Aspergillusflavus, 267,000; Afu—Aspergillus fumigatus, 1,000,000; Ani—Aspergillusniger, 458,750; Are—Aspergillus repens, 61,500; Ars—Aspergillusrestrictus, 38,000; Asy—Aspergillus sydowii, 875,000; Ate—Aspergillusterreus, 1,000,000; Ave—Aspergillus versicolor, 712,000;Apu—Aureobasidium pullulans, 961,250; Bci—Botrytis cinerea, culturesurface washing only; Ccl—Cladosporium cladosporioides, 371,250;Che—Cladosporium herbarum, 807,500; Csp—Cladosporium sphaerospermum,60,500; Cgl—Chaetomium globosum, culture surface washing only;Clu—Curvularia lunata, culture surface washing only; Eam—Eurotiumamstelodami, 29,750; Eni—Epicoccum nigrum, 5,750; Fcu—Fusarium culmorum,389; Fmo—Fusarium moniliforme, 1,300,000; Fox—Fusarium oxysporum,845,000; Fso—Fusarium solani, 8,750; Ftr—Fusarium tricinctum, 34,000;Gca—Geotrichum candidum, 1,200,000; Mec—Memnoniella echinata, 262,500;Mra—Mucor ramannianus, 26,750; Mve—Myrothecium verrucaria, 40,750;Pbr—Penicillium brevicompactum, 1,000,000; Pch—Penicillium chrysogenum,633,333; Pci—Penicillium citrinum, 1,000,000; Pex—Penicillium expansum,1,000,000; Pis—Penicillium islandicum, 521,250; Ppu—Penicilliumpurpurogenum, 30,000; Pro—Penicillium roqueforti, 466,250;Pva—Paecilomyces variotii, 535,000; Pve—Penicillium variabile,1,000,000; Rst—Rhizopus stolonifer, 481,000; Sal—Stachybotrys albipes,6,500; Sbi—Stachybotrys bisbyi, 6,250; Sbr—Scopulariopsis brumptii,47,500; Sch 1-14—Stachybotrys chartarum isolates 1-14, 71,750; 33,000;62,000; 89,250; 32,500; 29,500; 78,000; 36,250; 64,750; 66,250; 21,500;79,250; 43,500; 103,000 respectively; Scy—Stachybotrys cylindrospora,35,750; See—Stachybotrys echinata, 109,000; Ska—Stachybotryskampalensis, 2,000; Smi—Stachybotrys microspora, 27,000;Sne—Stachybotrys nephrospora, 22,500; Ssu—Stachybotrys subsimplex,2,000; Tha—Trichoderma harzianum, 1,000,000; Uch—Ulocladium chartarum,31,500; Wse—Wallemia sebi, 205,250.

DETAILED DESCRIPTION

Antibodies that selectively bind to antigens produced by fungi commonlyfound in indoor environments are disclosed. In some embodiments, amonoclonal antibody selectively binds an antigen produced byStachybotrys chartarum (Ehrenb.) Hughes (Syn. Stachybotrys atra Corda,Stachybotrys alternans Bonord), such as monoclonal antibody 9B4, whichselectively binds to the spores, but not the mycelium, of Stachybotryschartarum.

Abbreviations

-   -   ATCC: American Type Culture Collection    -   BSA: bovine serum albumin    -   CCB: carbonate coating buffer    -   CDR: complementarity-determining region    -   CSN: culture supernatant    -   DMEM: Dulbecco's Modified Eagle Medium    -   ELISA: enzyme-linked immunosorbent assays    -   ELISPOT: enzyme-linked immunospot assays    -   KLH: keyhole limpet hemocyanin    -   Mab: monoclonal antibody    -   PBS: phosphate buffered saline    -   PBST: phosphate buffered saline supplemented with 0.05% Tween 20    -   PCR: polymerase chain reaction    -   RAST: radioallergosorbent tests    -   RT: room temperature        Explanations of Terms

The following explanations of terms are provided to better illustratecertain features of the invention and to facilitate review of theembodiments described herein. Explanations of common terms also can befound in Goldsby, R. A. et al., Kuby Immunology, 4^(th) edition, W. H.Freeman & Co.: New York, 2000; Janeway, C. (Editor) and Travers, P.,Immunobiology, 5^(th) edition, Garland Pub: New York, 2001; and Harlow,E., and Lane, D., Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1998.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antibody” includes single or pluralantibodies and can be considered equivalent to the phrase “at least oneantibody.”

The term “or” refers to a single element of stated alternative elementsor a combination of two or more elements. For example, the phrase “amonoclonal or polyclonal antibody” refers to a monoclonal antibody, apolyclonal antibody, or both a monoclonal and a polyclonal antibody.

As used herein, “comprises” means “includes.” Thus, “comprising anantibody and antigen” means “including an antibody and antigen,” withoutexcluding additional elements.

Animal. A living multicellular vertebrate organism, including mammals,fish, and birds. A “mammal” includes both human and non-human mammals.Similarly, the term “subject” includes both human and veterinarysubjects.

Antibody. Serum protein formed in response to immunization. Animmunoglobin molecule that has an amino acid sequence by virtue of whichit interacts with the antigen that induced its synthesis or with relatedantigens.

Antigen. A substance capable of inducing a specific humoral or cellularimmune response and capable of reacting with the products of thatresponse (i.e., specific antibodies) or which can be bound by preformedantibodies. Those antigens capable of inducing antibody production arecalled “immunogens.” Antigens can be soluble molecules such as proteins,lipids, carbohydrates, or nucleic acids; or particulates such asbacteria, virus, or fungi, as whole cells, whole particles, or fragmentsthereof. Antigens can have complex surfaces and can express multipleunique antigenic determinants or epitopes. An antigen expressingdifferent independent epitopes is said to be multi-determinant, while anantigen expressing multiple copies of a given epitope is said to bemultivalent.

Antigens of Stachybotrys chartarum can be intracellular or secreted intothe environment by viable propagules, or released by disintegratingnon-viable propagules. Examples of such antigens include structuralproteins, cell-surface proteins, secreted proteins, and fragmentsthereof.

Binding reaction. Occurs when a binding agent (such as an antibody)interacts with its target (such as an antigen). In some cases, a bindingreaction occurs when two structurally and/or energetically complementarymolecular surfaces react to form a ligand-receptor complex withcharacteristic specificity and affinity. For example, a monoclonalantibody can undergo a binding reaction with its cognate antigen to forman antigen-antibody complex, also called an immune complex. Thus, abinding reaction can be referred to as a “complexing reaction.”

An agent that “selectively” binds to a particular target exhibits somepreference for its target over other similar targets. Some antibodies(both monoclonal and polyclonal) can discriminate between closelyrelated epitopes and also can distinguish between different antigens, ifthose antigens express epitopes that are not shared by other antigens.As one non-limiting example, Mab 4E12 reacts with an epitope that isexpressed by 15 out of 52 (29%) fungal species tested. See Table 2.

An agent that “specifically” binds to a particular target bindssubstantially only to a defined target. As used herein, a specificbinding agent includes both monoclonal and polyclonal antibodies. As onenon-limiting example, monoclonal antibody 9B4 specifically binds to anantigen of Stachybotrys chartarum spores that is not found in eitherStachybotrys chartarum mycelium or spores or mycelia of other fungalspecies tested, including other Stachybotrys species. See Table 2.

Epitope. The local portion of an antigen recognized by a correspondingantibody, also called an “antigenic determinant” or “antibody bindingsite.” An epitope is any antigenic determinant on an antigen to whichthe paratope of an antibody binds. Epitopic determinants can consist ofchemically active surface groupings of molecules, such as amino acids orsugar side chains, and can have specific three-dimensional structuralcharacteristics and specific charge characteristics.

Fungus. Living, single-celled and multicellular organisms belonging toKingdom Fungi. Characterized by a lack of chlorophyll and presence ofchitinous cell walls in some species. May be multinucleated.

Hybridoma. A cell line or culture that secretes a homogenous populationof monoclonal antibodies. Hybridomas are hybrid cells resulting from thefusion of a myeloma (tumor cell), which confers immortality, and anantibody-producing cell, which confers antibody specificity onto thehybridoma. One specific, non-limiting example is the hybridoma capableof producing Mab 9B4 that was deposited on Aug. 9, 2002 with theAmerican Type Culture Collection (ATCC, Manassas, Va.) under ATCCaccession number PTA-4582.

Isolated. An “isolated” biological component—such as a molecule,antibody, nucleic acid, or cellular organelle—has been substantiallyseparated or purified away from other biological components of theorganism in which the molecule, antibody, nucleic acid or cellularorganelle naturally occurs. The term “isolated” thus encompassesantibodies purified by standard protein purification methods. The term“isolated” also embraces antibodies prepared by recombinant expressionin a host cell and chemically synthesized antibodies.

Label. A chemical group attached to an antigen, antibody or othermolecule to facilitate its detection. An antigen, antibody or othermolecule may be labeled for direct detection, such as by methodsdescribed in Harlow and Lane (1998). Suitable labels include, but arenot limited to, an enzyme, a radiolabel, a fluorescent label, acolorimetric label, a luminescent label, or biotin, and may be chosenbased on such functions as availability of detection methods,availability of equipment, affinity of the label and labeled molecule,or degree of interference on the activity of the labeled molecule.

Monoclonal antibody (Mab). Identical antibodies derived from a singleplasma cell, antibody-secreting cell, or a clone of cells thereof, thatdemonstrate specific affinity for certain antigen epitopes. A monoclonalantibody can be a protein synthesized in pure form by a singlepopulation of cells, such as being secreted by a hybridoma. The term“oligoclonal antibodies” or “antibody cocktail” refers to apredetermined mixture of distinct monoclonal antibodies. Also includedis any antibody derivative based on any Mab disclosed herein, such asproteolytic fragments including, but not restricted to, Fab, Fab′,F(ab′)₂, Fv, Fd, or Fb fragments or any recombinant reagent based on thenucleotide or amino acid sequences of the immunoglobulin heavy or lightchains.

In contrast, polyclonal antibodies, or immune sera, are a mixture ofdifferent antibodies with specificities for independent epitopesexpressed by an antigen. Polyclonal antibodies are thus a diversepopulation of antibodies secreted by all antigen-reactive plasma cellsduring an immune response.

Nucleotide. A monomer composed of a sugar moiety, phosphate group, andan amine base, such as a pyrimidine, purine, or synthetic analogsthereof. A nucleotide is one monomer in a polynucleotide, such as anucleic acid molecule. A nucleic acid sequence refers to the sequence ofbases in a polynucleotide.

Polyclonal antibody. A mixture of different antibodies purified fromhyperimmune serum. Polyclonal antibodies can be produced by severaldifferent methods, such as those described in Green et al., in:Immunochemical Protocols Manson, M. (Ed.), Humana Press, 1992, pp. 1-5;and Coligan et al., in: Current Protocols in Immunology, Coligan, J. E.et al. (Eds), Wiley and Sons, New York, 1992, section 2.4.1.

Polypeptide. A polymer in which the monomers are amino acid residuesjoined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. A “protein” may be composed of a single polypeptidemolecule, or may be composed of plural polypeptide subunits.

The terms “polypeptide” and “protein” encompass any amino acid sequenceand include modified sequences, such as glycoproteins. The term“polypeptide” encompasses naturally occurring proteins, as well as thosethat are recombinantly or synthetically produced.

A “fragment” of a polypeptide refers to a portion of a polypeptide thatexhibits at least one useful epitope. The term “functional fragments” ofa polypeptide refers to all fragments of a polypeptide that retain anactivity of the polypeptide, such as the ability to bind a particularfungus or range of fungi. Biologically functional fragments, forexample, can vary in size from a polypeptide fragment as small as anepitope capable of binding an antibody molecule to a large polypeptidecapable of participating in the characteristic induction or programmingof phenotypic changes within a cell.

Therapeutic agent. A substance that demonstrates some therapeutic effectby restoring or maintaining health, such as by alleviating the symptomsassociated with a disease or physiological disorder, or interfering witha pathophysiological process that leads to a disease or its progression.In some instances, the therapeutic agent is an antibody, a chemical orpharmaceutical compound, or a nucleic acid molecule. A therapeutic agentcan be an antifungal agent—an agent effective against fungal infectionsand destructive to fungi or capable of suppressing their growth orreproduction—or an antitoxin. As one non-limiting example, thetherapeutic agent is an antitoxin to the toxin produced by Stachybotryschartarum suitable for administration to humans.

Antibodies

Polyclonal and monoclonal antibodies against various fungal antigens maybe produced using cell lines, such as hybridomas. In some embodiments,an antibody is produced by repeatedly immunizing a mammal, such as amouse, with a target antigen until an immune response in the mammal isdetected. For example, antibody production in a mouse can be verified bycollecting and analyzing serum samples by ELISA. After the mammal issacrificed, a single cell suspension is prepared from its spleen.Individual splenocytes, including many antigen-reactive plasma cells,are then immortalized by cell fusion with a myeloma cell line. Thesuccessfully fused cells (hybridomas) are cloned by dilution withaliquots placed in wells of a microtiter plate for culture growth, theclones are screened for production of antibody to the antigen, andantibody is isolated from the positive cloned hybridomas. For example,hybridomas may be aliquoted into wells of 96-well ELISA plates and,after about 10-14 days of cultivation in selective medium culture,supernatants of individual wells with cell growth are tested for thepresence of target antigen-specific antibodies using a screeningtechnique; cells of positive wells are then cloned twice by limitingdilution in order to ensure monoclonality of the resulting hybridoma.Techniques for producing and isolating monoclonal antibodies aredescribed in detail in various references, such as in Monoclonalantibodies—Production, Engineering and Clinical Applications, Ritter, M.A., and Ladyman, H. M. (Eds.). Cambridge University Press, 1995; BasicMethods in Antibody Production and Characterization, Howard, G. C., andBethell, D. R. (Eds.), CRC Press, 2001; Diagnostic and TherapeuticAntibodies, George, A. J. T., and Urch, C. E. (Eds.), Humana Press,2000; and Harlow, E., and Lane, D. (Eds.), 1998.

Fungal antigens used to stimulate antibody production can be obtainedfrom whole cells; fragments of cells; surface washings; or secreted,released, or extracted cellular molecules. For example, fungal cells(from spores or mycelia) can be solubilized and injected into an animal.Antibodies also can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or protein used to immunize an animal may be derived fromtranslated cDNA or chemical synthesis and can be conjugated to a carrierprotein, if desired. Commonly used carrier proteins include keyholelimpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), andtetanus toxoid.

Any animal having an immune system can be used in antibody production,such as rats, hamsters, pigs, felines, and primates, includingchimpanzees, baboons, and humans.

As described in Example 1, a hybridoma may be produced by fusinghyperimmune spleen cells with SP2/0-AG14 myeloma cells using standardtechniques, though monoclonal antibodies may be produced using othermyelomas.

Once a cell line is produced, that cell line is cultured for a period oftime, and each culture supernatant is screened against a target fungusto determine if the cell line produces a reactive antibody. A number ofimmunoassay techniques can be used to screen the culture supernatants,such as competitive and non-competitive homogenous and heterogenousenzyme-linked immunosorbent assays (ELISA) as symmetrical orasymmetrical direct or indirect detection formats; enzyme-linkedimmunospot assays (ELISPOT); radioallergosorbent tests (RAST);fluorescent tests, such as used in fluorescent microscopy and flowcytometry; Western, grid, dot or tissue blots; dip-stick assays; halogenassays; or antibody arrays. See, e.g., O'Meara, T. and Tovey, E.,Clinical Reviews in Allergy and Immunology, 18:341-95 (2000); Sambrooket al., (2001), Appendix 9; Simonnet, F., and Guilloteau, D., in:Methods of Immunological Analysis, Masseyeff, R. F., et al. (Eds.), VCH,New York, 1993, pp. 270-388. Other analytical techniques include immunecomplex-based assays such as immunodiffusion, precipitation,coagulation, agglutination, and hemagglutination techniques, as well astests based on turbidimetry or nephelometry.

A positive reaction indicates that the antibody produced by a particularcell line binds to or complexes with an antigen produced by a particulartarget fungus. Positive cultures are subcloned and developed by in vitroculture over long periods of time into stable cell lines that produceantibodies.

Monoclonal antibodies can be isolated from hybridoma cultures by avariety of methods, including affinity chromatography with Protein-ASepharose, size-exclusion chromatography, and ion-exchangechromatography. Other purification methods include binding to andelution from a matrix containing the antigen to which the antibodieswere raised that is bound to some substrate. See, e.g., Coligan et al.,sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3 (1992); Barnes et al.,in: Methods in Molecular Biology, Vol. 10, Humana Press 1992; pp.79-104; Coligan et al., Current Protocols Immunology, WileyInterscience, 1991, Unit 9.

Particular antibodies can be selected based on the fungal antigens towhich they bind. Generally, an animal is immunized with a fungalantigen, which may itself be pre-selected, and antibody-producing cellsfrom that animal are used as a source for producing monoclonal orpolyclonal antibodies. The resulting antibodies are isolated andscreened for binding activity with various fungi, and particularantibodies are selected based on their binding patterns. For example,monoclonal antibodies that selectively or specifically bind Stachybotryschartarum antigens can be produced by hybridomas resulting fromantibody-producing cells obtained from animals immunized withStachybotrys chartarum antigens, such as antigens from Stachybotryschartarum spores, mycelia, or fragments or molecules thereof. Examples1-3 below describe in detail how to produce a monoclonal antibody (9B4)that specifically binds to an antigen of Stachybotrys chartarum spores.However, the techniques described in Examples 1-3 can be modified toproduce other monoclonal antibodies that, like 9B4, specifically bind anantigen of Stachybotrys chartarum spores. Additionally, the techniquescan be modified to produce antibodies that specifically (or selectively)bind an antigen of another fungal species or part of a fungus.

Antibodies can be multiplied in vitro and in vivo. Multiplication invitro can be carried out in suitable culture media, such as Dulbecco'sModified Eagle Medium or RPMI 1640 medium, optionally supplemented by amammalian serum, such as fetal calf serum, or trace elements andgrowth-sustaining supplements, such as normal mouse peritoneal exudatecells, spleen cells, thymocytes, or bone marrow macrophages. Productionin vitro provides relatively pure antibody preparations and can bescaled to yield large amounts of the desired antibodies. Large scalehybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture.

Multiplication in vivo can be carried out by injecting cell clones intomammals histocompatible with the parent cells, such as syngeneic mice,to cause growth of antibody-producing tumors. Optionally, the animalscan be primed with a hydrocarbon, especially oils, such as pristane(tetramethylpentadecane), prior to injection. After one to three weeks,the desired antibody is recovered from the body fluid of the animal.

An antibody also can be derived from a non-human primate antibody. Forexample, techniques for raising therapeutically useful antibodies inbaboons can be found in Goldenberg et al., International PatentPublication WO 91/11465, 1991, and Losman et al., Int. J. Cancer 46:310(1990).

Antibodies can be derived from a human monoclonal antibody. Suchantibodies are obtained from a transgenic vertebrate organism, such as amouse, that has been genetically engineered to produce a human antibodyin response to antigenic challenge. Elements of the human heavy andlight chain loci are introduced into strains of these organisms derivedfrom embryonic stem cell lines that contain targeted disruptions of theendogenous heavy and light chain loci. The transgenic organisms cansynthesize human antibodies specific for human antigens, and theorganisms can be used to produce human antibody-secreting hybridomas.Methods for obtaining human antibodies from transgenic organisms, suchas mice, are described by Green et al., Nature Genet. 7:13 (1994);Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immunol.6:579 (1994).

For human applications, animal-derived antibodies can be geneticallymanipulated or engineered to reduce their immunogenicity. For example,several chimeric or CDR-grafted humanized antibodies have been evaluatedin clinical trials. See, e.g., Gavilondo, J. V., et al., TheImmunologist 8:58-65 (2000); Glennie, M. J., and Johnson, W. M.,Immunology Today, 21:403-10 (2000). Alternatively, human antibodies canbe produced from human peripheral donor lymphocytes or generated fromimmunized transgenic mice known as xenomice or translocus mice, whichexpress human immunoglobulin genes instead of the constitutive mousegenes. See, e.g., Jakobovits, A., Advanced Drug Delivery Reviews31:33-42 (1998); Nicholson, I. C., et al., Journal of Immunology,163:6898-6906 (1999); Gallo, M. L., et al., European Journal ofImmunology 30:534-540 (2000).

Another approach for producing human antibodies is based on thereconstitution of irradiated and scid bone marrow radioprotected micewith human peripheral blood mononuclear cells. Such mice, called trimeramice, can be immunized and used as a source of specific human plasmacells to produce antibodies. See, e.g., Reisner, Y., and Dagan, S.,Trends in Biotechnology 16:242-46 (1998); Eren, R., et al., Hepatology32:588-596 (2000).

Antibodies also can be derived from human antibody fragments isolatedfrom a combinatorial immunoglobulin library. See, for example, Barbas etal., in: Methods: A Companion to Methods in Enzymology, Vol. 2, page 119(1991); Winter et al., Ann. Rev. Immunol. 12:433 (1994). Cloning andexpression vectors useful for producing a human immunoglobulin phagelibrary can be obtained, for example, from Stratagene Cloning Systems(La Jolla, Calif.).

In addition to production by hybridomas, antibodies can be produced bymethods based on recombinant DNA technologies, such as phage display ofcombinatorial libraries or genetic engineering. See, e.g., Little, etal., Phage Display: A Laboratory Manual, Barbas, C. F. (Ed.), ColdSpring Harbor Laboratory Press, 2001.

An antibody can be labeled for direct detection, such as by methodsdescribed in Harlow and Lane (1998). Suitable labels include, but arenot limited to, an enzyme, a radiolabel, a fluorescent label, acolorimetric label, a luminescent label, or biotin, and can be chosenbased on the method of detection available to the user or otherconsideration.

A monoclonal antibody can be conjugated with a desired agent, such as atherapeutic agent or metal chelate. These agents can be conjugatedchemically, using a chemical reaction that is dependent upon thefunctional groups present on the agent to be conjugated, produced byphotocrosslinking, or generated using other techniques. See, e.g., Best,U.S. Pat. No. 5,082,928 and references contained therein.

The antibodies disclosed herein include intact molecules, and fragmentsthereof, capable of binding the epitopic determinant of their cognateantigens. Antibody fragments can be produced by a variety of methods,such as by the targeted proteolysis methods described in Harlow and Lane(1998), or recombinant DNA techniques. See, e.g., Little, M., et al.,Immunology Today 21:364-370 (2000). Alternatively, the nucleic acidsequences encoding the heavy and light chains of an antibody can becloned using universal but immunoglobulin-specific primers, sequenced,and produced in vitro using a variety of expression systems. See, e.g.,Ladiges, W., and Osman, G. E., in: Basic Methods in Antibody Productionand Characterization, Howard, G. C., and Bethell, D. R. (Eds.), CRCPress, Boca Raton, 2001, pp. 169-91.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by in vivo expression of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by various methods, such as the methods described inGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein. See also Nisonhoff et al., Arch. Biochem. Biophys.89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al.,Methods in Enzymology, Vol. 1, page 422, Academic Press (1967); andColigan et al. (1992) at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides, also known as“minimal recognition units,” can be obtained by constructing nucleicacid sequences encoding the CDR of an antibody of interest. Such nucleicacid sequences are prepared, for example, by using the polymerase chainreaction (PCR) to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: aCompanion to Methods in Enzymology, Vol. 2, page 106 (1991).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques also canbe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

It is also possible to use anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

Methods of Using Antibodies

Antibodies disclosed herein can be used as experimental research tools,or for diagnostic, prophylactic, or therapeutic purposes in a clinicalsetting. Additionally, these antibodies can be used to detect and/ormonitor fungi in plants, animals, or the environment according to theirindividual cross-reactivity profiles, as summarized in Table 2 below.For example, Mab 9B4 can be used for species-specific detection ofStachybotrys chartarum spores.

In some embodiments, an antibody is used to detect the presence of orexposure to a fungus within an original or processed sample obtainedfrom the environment, a plant, or an animal. Samples obtained from theenvironment include soil, water, dust, bulk samples (e.g., buildingmaterial or furniture), air, and other reservoir samples, such as animalfeed, collected in an indoor or outdoor environment. For example, airsamples may be collected in a ventilation duct of a building. Samplesobtained from a plant or animal may include a cell, tissue, biopsy,extracted gall, secretion, or fluid from the subject. Regarding animalsubjects, the sample may be obtained from a bodily fluid (e.g., urine,lymph, blood, serum, plasma, cerebrospinal fluid, saliva, and swipe orsmear samples from mucosal surfaces) or gastric contents (i.e., thecontents of the gastrointestinal system, including stomach contents,gastrointestinal biopsy, and feces). In particular embodiments, thesample is obtained from a lung of an animal, such as by biopsying lungtissue, or collecting a bronchial, bonchioalveolar, nasal, or sputumsample.

The sample in its entirety can be taken solely from the subject, such asby probing or scraping, or can be collected through the addition of someother substance or compound. For example, a sample can simply be bloodor urine collected from a subject animal and aliquotted for contactingwith the monoclonal antibody. As an alternative example, a sample can beobtained from a plant, animal, or some environmental surface by surfacewashings with sterile water, a dedicated extraction buffer, or othersuitable fluid.

A sample can be analyzed directly, extracted before analysis, orexpanded in volume by the addition of a suitable solvent. For example, asample of fungal spores can be collected using a screen or trap placedin a ventilation duct of a building; the spores collected from theairflow through the duct can be fragmented and suspended in solution. Ifthe type or volume of sample obtained is not sufficient for screeningwith the antibody, the sample can be expanded by the addition of asuitable organic or inorganic solvent, or can be expanded by culturingin a growth medium suitable for culturing fungi suspected of being inthe sample.

The sample is contacted with an amount of monoclonal antibody, and thesamples are screened to detect a monoclonal antibody complexing with anantigen, such as detecting the binding reaction between the antibody andantigen. Detection of an antibody-antigen complex or binding reactionindicates that the sample was exposed to or contacted by a fungus havingan antigen to the antibody. In some instances, the fungus (spores,mycelium, or both) is still present in the sample. In other instances,the fungus has degraded, disappeared from, or been eliminated from thesample, leaving behind antigenic components.

For example, labeled 9B4 monoclonal antibodies can be used to detectantigen of Stachybotrys chartarum spores, thus indicating that thesample was exposed to Stachybotrys chartarum spores. In someembodiments, the antibody is labeled with an environmentally sensitivelabel, such as a fluorescent label, so that its signal changes uponbinding to antigen. In other embodiments, antigen in the sample isimmobilized on a surface prior to introduction of the labeled antibody,and the amount of the signal, corresponding to the amount of boundlabeled antibody, correlates to the amount of antigen in the sample. Instill other embodiments, antigen is captured by immobilized unlabeledfirst antibody, after which a labeled second antibody is introduced tobind to the captured antigen and produce a signal in proportion to theamount of captured antigen.

If the sample was obtained from the environment, such as an air sampleobtained from the ventilation duct of a building, then the environmentcan be searched for the presence of Stachybotrys chartarum. For animalsubjects, particularly people and domesticated animals, a determinationof Stachybotrys chartarum exposure can aid in diagnosing disease anddeveloping treatment regimens.

In addition to using an antibody to detect an antigen, the antibody canbe used to isolate its corresponding antigen, and the antigen can thenbe used to detect the presence of that antibody or other antibodies tothat antigen. For example, an antibody can be used to affinity purifyits cognate antigen, which can then be used in competition assays toquantify homologous antigen in a sample obtained from a plant, animal,or the environment.

In such embodiments, a monoclonal or polyclonal antibody is isolated andpurified and used in an immunoprecipitation procedure to isolate thecorresponding antigen from a mixture of soluble molecules, such as asuspension of lysed cells. Exemplary immunoprecipitation procedures canbe found in Harlow and Lane (1998), chapter 7. Once the antigen has beenisolated, it can be analyzed by determining characteristics such asmolecular weight, sequence, and three-dimensional structure. Suchcharacterization is not necessary for subsequent uses of the antigen,however. Once isolated, the antigen is used to screen a sample to detectthe presence of a cognate antibody. In some embodiments, the antigen islabeled, and the labeled antigen is used to detect cognate antibody inthe same way that labeled antibody is used to detect antigen (describedabove). In other embodiments, the antigen is immobilized, and antibodyis captured and then detected, for example, by ELISA.

Purified antigen can be used to identify the presence of a correspondingantibody in an animal subject, thus establishing that the animal subjectwas exposed to a fungal-antigen recognized by the antibody. For example,monoclonal antibody 9B4 can be used to isolate and immunoprecipitate itscorresponding antigen (see Example 4), and this antigen can be labeledand used in a solid-phase or competition assay to screen a blood sampleobtained from a human patient for the presence of antibody to thatantigen, the presence of antibody indicating that the person was exposedto Stachybotrys chartarum.

Additionally, antibodies also can be utilized to screen phage displayedor synthetic expression libraries for peptides or mimotopes that may beused as functional single-epitope antigens. Such mimotopes can beconsidered species-specific, if the template antibody used for screeningis species-specific. Thus, by reducing antigens to the size of epitopicpeptides, potential ambiguities due to other epitopes expressed by thefull length parent molecule can be avoided. For example, fungi have beenfound to share epitopic determinants and to cross-react widely. Thus,the production of mimotopes for Mab 9B4 can allow the development ofdefinitive exposure measurement techniques for animal subjects. Theantibodies also can be used in quality assurance, standardization, andcharacterization of fungal extracts to be used in clinical practice orresearch.

The disclosed antibodies can be used in a variety of immunometricassays, including radiolabeled, enzyme, fluorescence, precipitation,agglutination, coagulation, Western blot, grid blot, tissue blot, dotblot, chemiluminescence, light-scattering, electrochemical, dip-stick,or biosensor assays. See, e.g., Principles aid Practice of Immunoassays,Price, C. P. and Newman, D. J. (Eds.), Stockton Press, 1997; TheImmunoassay Handbook, 2^(nd) Edition, Wild, D. (Ed.), Nature PublishingGroup, London, 2001. In general, an antibody can be used as a labeledprimary reagent in a direct assay or as an unlabeled reagent to bedetected by a secondary developing antibody conjugate, such as labeledanti-mouse antibody, in an indirect assay. Additionally, an antibody canbe used in a competition assay to detect an antigen or antibody in asample. For example, antigen in a sample extract can be captured by anunlabeled antibody immobilized on the surface of an ELISA well and thendetected by a labeled antibody of the same or different kind and/orspecificity. Alternatively, the sample can be suspended in a buffer andmixed directly with an antibody, thus allowing the antibody to form animmune complex with its antigen. The reduction of free antibody due tocomplex formation can then be determined in a second step, based onsolid-phase ELISA with purified antigen, by comparing the relativereactivity of free residual antibody left over after sample incubation(sample reactivity) to that of the same antibody when not mixed with thesample (reference reactivity). The ratio of sample to reference antibodyreactivity will be inversely proportional to the amount of antigen inthe sample.

In other embodiments, an antibody is used for the detection of fungalpropagules on collection surfaces, such as transparent adhesive tapes orair filters, using fluorescent microscopy, a halogen assay, or someother detection method. See, e.g., O'Meara, T., and Tovey, E., ClinicalReviews in Allergy and Immunology 18:341-95 (2000). For example,fluorescently labeled Mab 9B4 can be used to detect spores ofStachybotrys chartarum. In a halogen assay, air particles are collectedvia an adhesive tape, and antigens are transferred by elution to alaminated protein-binding membrane. Particle-characteristic bindingpatterns, such as halos or spots, are formed on the membrane, and thepatterns are visualized for counting by antibody-mediated particlestaining using insoluble enzyme substrates.

Antibodies also can be used to test the effectiveness of an agent incomplexing with a fungal antigen. In some embodiments, a monoclonalantibody is mixed with its antigen and an agent. The antigen can beborne on a fungus or a portion of the fungus, or purified apart from thefungus. The mixing of the antibody, antigen, and agent can beaccomplished in vitro or in vivo. An agent that complexes with the sameantigen as the antibody will inhibit the antibody-antigen bindingreaction. Therefore, a decrease in antibody-antigen binding in thepresence of the agent compared to some reference standard (e.g.,background fluorescence) or control (e.g., antibody-antigen binding inthe absence of the agent) indicates that the agent may interact with theantigen. One exemplary method of assessing antibody-antigen binding isto analyze the kinetics of the binding reaction and determine a rate ofbinding. Other exemplary methods include determining the total amount ofantibody-antigen binding at equilibrium.

In other embodiments, an antibody is used in histochemistry or imageanalysis to visualize, identify, localize or quantify specificmolecules, such as being used in proteomics to describe proteinexpression patterns for signal transduction or in metabolic pathways. Inclinical applications, an antibody may be used for environmental orpatient monitoring in allergy or toxicology applications, for immunesuppression and tolerance induction in autoimmunity and transplantation,or for the treatment of infections or tumors. For example, a Mabconjugated to a toxin, prodrug, or radioisotope can be used inantibody-guided therapy for disease or infection. Bamias, A., andEpenetos, A. A., in: Monoclonal Antibodies—Production, Engineering andClinical Applications, Ritter, M. A. and Ladyman, H. M. (Eds.),Cambridge University Press, Cambridge, 1995, pp. 222-46. As anotherexample, a Mab can be used as a surrogate antigen in anti-idiotypetherapy, or an antibody can be genetically engineered as an antigenizedantibody to express an antigen in a complementarity-determining region.See, e.g., Polonelli, L., et al., in: The Antibodies—Volume 4, Zanetti,M. and Capra, J. D. (Eds.), Harwood Academic Press, Amsterdam, TheNetherlands, 1997, pp. 143-66; Bona, C. A., Nature Medicine 4:705-706(1998); and Vogel, M., et al., Journal of Molecular Biology 298:729-735(2000).

An antibody also can be used to direct an agent—such as an antibiotic,therapeutic agent, or metal chelate—to a fungus. In some embodiments, anantibody is conjugated with the agent and the conjugated agent is thenable to target a particular fungus or several different fungi, dependingon which antibody is used. The antibody portion of such a conjugate maycomplex with antigens found on a fungus and, thus, bring the agent intoeffective proximity of the fungus. For example, a fungi static agenteffective against Stachybotrys chartarum conjugated with the 9B4monoclonal antibody would selectively target Stachybotrys chartarumspores. Moreover, if the antigen has been secreted from the fungus, orif the fungus has disintegrated, the antibody-agent complex can bind theantigen and prevent that antigen from interacting with other molecules.

In particular embodiments, an antibody-agent conjugate, such as aMab-fungistatic or Mab-fungitoxic compound, is introduced into aparticular locus of the environment. For example, conjugates of Mab 9B4can be applied to particular sites within a building known or suspectedto be contaminated by Stachybotrys chartarum.

In other embodiments, an antibody-agent conjugate, such as aMab-fungistatic or Mab-fungitoxic compound, is introduced into a subjectanimal or plant for diagnostic, prophylactic, therapeutic, or otherpurposes. For example, Mab 3B2 conjugates can be used to targettherapeutic agents or prodrugs to fungal spores or mycelia in a subjectknown or suspected to be exposed to any of Aureobasidium pullulans,Cladosporium cladosporioides, Cladosporium herbarum, Cladosporiumsphaerospermum, Memnoniella echinata, Myrothecium verrucaria,Stachybotrys albipes, Stachybotrys bisbyi, Stachybotrys chartarum,Stachybotrys cylindrospora, Stachybotrys microspora, Stachybotrysnephrospora, or Stachybotrys subsimplex.

Some embodiments include a kit or article of manufacture that includesan antibody. In these embodiments, an antibody is stored within acontainer capable of storing the antibody for its shelf life. Thecontainer can be made of any suitable material such as plastic or otherpolymer, glass, metal, or the like. Printed instructions and/or aprinted label indicating a method of use for the antibody, such asinstructions describing how the antibody can be used to detect fungi,can be associated with the container. The instructions and/or label mayprovide information regarding the use of the antibody for any of thepurposes in accordance with the methods set forth herein and can beassociated with the container by being adhered to the container oraccompanying the container in a package. Additionally, the container caninclude a feature or device for using the antibody. In certainembodiments, the article of manufacture includes, packaged together, avessel containing the antibody and instructions for use of the antibodyfor detecting a fungus.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Preparation of a Parent Hybridoma

Spores of Stachybotrys chartarum were harvested from sporulating,air-dried 7-day old culture plates containing 5 ml of malt extract brothagar. Two isolates of Stachybotrys chartarum, one toxigenic and onenon-toxigenic, were used as an antigen mixture. Spores were collectedfrom sporulating cultures with 1 g of glass beads (0.45-0.5 mm, B. BraunBiotech International, Melsungen, Germany), which were gently shakenacross fungal colonies. This allowed ample spores to attach to the beadsand also allowed easy recovery of the spores for ELISA analysis bysimply shaking the beads in a 50 ml tube containing carbonate coatingbuffer (CCB).

Three, 12-week old female BALB/c mice, obtained from the animal facilityof the National Institute for Occupational Safety and Health, wereimmunized intraperitoneally approximately every two weeks for severalmonths. For immunogen preparation, harvested spores were suspended in 10mM phosphate buffered saline (PBS, Sigma, St. Louis, Mo.), counted, andwashed twice by centrifugation at 15 g for 5 min in PBS supplementedwith 0.05% Tween 20 (PBST). The final pellet was re-suspended in PBS,and 1 ml aliquots of the spore suspension were supplemented with 1 g ofglass beads (0.45-0.5 mm, B. Braun Biotech International, Melsungen,Germany). Spores were then mechanically homogenized using a bead beaterfor three, 2 min intervals, while being kept on ice for 2 min betweenintervals. The aliquots were combined, and particulate cell debris wascollected in PBS after two washes by centrifugation at 15 g for 10 min.Mice were immunized with the particulate fraction at concentrationsequivalent to the spore numbers shown in Table 1.

TABLE 1 Immunization of mice Immunized Spores per Treatment Mouse 1^(st)immunization   1 × 10⁶ 2^(nd) immunization 0.5 × 10⁶ 3^(rd) immunization2.5 × 10⁶ 4^(th) immunization   5 × 10⁶ 5^(th) immunization 1.5 × 10⁶6^(th) immunization   2 × 10⁶ - Mouse 1   1 × 10⁶ - Mice 2 and 3 7^(th)immunization   2 × 10⁶ - Mouse 2   1 × 10⁷ - Mouse 3

Hyperimmune mice were sacrificed and single cell suspensions wereprepared from aseptically removed spleens. Ladyman, H. M., and Ritter,M. A., in: Monoclonal Antibodies—Production, Engineering and ClinicalApplication, Ritter, M. A. and Ladyman, H. M. (Eds.), CambridgeUniversity Press, Cambridge, UK, 1995, pp. 429-66. Splenocytescontaining antigen-reactive plasma cells were fused with SP2/0-AG14myeloma cells (Cat# CRL-1581, ATCC, Manassas, Va.) using polyethyleneglycol 1500 (Boehringer Mannheim, Germany) as fusogen. Yokoyama, W. M.,Current Protocols in Immunology, Supplement 13:2.5.1 to 2.5.17 (1991).The fusion product of each individual fusion was suspended in 100 mlselective medium (Dulbecco's Modified Eagle Medium (DMEM), LifeTechnologies, Rockville, Md.) supplemented with 1 mM Pyruvate (LifeTechnologies, Rockville, Md.), 100 units/ml penicillin, 100 μg/mlstreptomycin, 0.292 mg/ml L-glutamine (Life Technologies, Rockville,Md.), 10% FCS (HyClone, Logan, Utah), 100 μM sodium hypoxanthine (LifeTechnologies, Rockville, Md.)), 0.57 μM azaserine (Sigma, St. Louis,Mo.) and 100 units/ml IL-6 (Life Technologies, Rockville, Md.). Cellswere seeded at 100 μl/well in 96-well tissue culture plates forincubation at 37° C. in 10% CO₂ in air. Cells were maintained in DMEMwithout azaserine, and culture supernatants were screened for reactivityto spores of Stachybotrys chartarum. Clones of positive wells werecloned twice by limiting dilution to ensure monoclonality and bulk grownin tissue culture plates to provide culture supernatant for Mabcharacterization. Stable clones were cryoprotectively stored in liquidnitrogen. Fusion 1 resulted in the production of Mabs 3B2 (IgM) and 10A5(IgM), fusion 2 generated Mabs 1D4 (IgM) and 9B4 (IgG₁), and fusion 3produced Mabs 9F9 (IgM) and 4E12 (IgM).

Example 2 Development of a Detection ELISA and Characterization ofMonoclonal Antibodies

Mabs produced in Example 1 were tested for cross-reactivity by ELISAagainst mycelial extracts of 12 isolates of S. chartarum (FIG. 2) and 29related and unrelated fungi commonly found in indoor environments (FIG.4). Mabs also were tested against spores of 14 isolates of S. chartarumand eight other Stachybotrys species (FIG. 1) and 43 other fungicommonly found in indoor and outdoor environments (FIG. 3). ELISA platewells were coated with mycelial extracts at 10 μg/ml protein or withspores at concentrations shown with the species code abbreviations.

Spores were harvested as described in Example 1, and mycelial extractswere prepared in liquid culture using spores as seeding inoculum.Sporulating slant cultures were washed with 2 ml of PBS, which was thentransferred to flasks containing 200 ml of Malt Extract Broth. Flaskswere incubated at room temperature (RT) and rotated at 200 rpm. Myceliumwas harvested after 10-11 days using a fine nylon mesh, and aliquotswere stored at −20° C. until further processing. For extraction,mycelium was washed in 50 ml carbonate/bicarbonate coating buffer (CCB:60 mM Na₂CO₃, 140 mM NaHCO₃, pH 9.6), shaken briefly, and pelleted at 15g for 10 min before being sonicated in 25 ml of CCB while kept on icefor 2 min. The sonicate was cleared by centrifugation at 15 g for 10 minbefore being centrifuged at 40,000 g for 20 min. The supernatant wasstored at −20° C. until used for cross-reactivity tests.

For ELISA analysis, wells of PolySorp ELISA plates (Nalge Nunc,Naperville, Ill.) were coated with a 100 μl suspension of spores ormycelium and incubated in a moist chamber at RT. After overnightincubation, wells were rinsed with three 10 min PBST-washes beforeunoccupied binding sites in the wells were blocked with 200 μl of PBSTcontaining 1% nonfat dry milk powder (Kroger, Cincinnati, Ohio) for 1 hat RT. Plates were washed and incubated for 1 h at 37° C. with 100 μl ofMab culture supernatant (CSN) diluted 5 times with PBST. After washing,bound Mab was detected with a 1/5000 PBST-diluted goat anti-mouseantibody conjugated to biotin (JacksonImmuno Research, West Grove, Pa.)by incubation at 37° C. for 1 hour. After washing, bound biotin waslabeled with a 1/5000 dilution of alkaline phosphatase-conjugatedstreptavidin (JacksonImmuno Research, West Grove, Pa.) in PBST byincubation for 1 hour at 37° C. Excess reagent was washed out, and theoptical density (OD) at 405 nm was determined using an ELISA reader(Bio-Tek Instruments, Winooski, Vt.) after incubating the substratep-nitrophenyl phosphate (Sigma, St. Louis, Mo.) at 0.5 mg/ml in 100 mMdiethanol-amine buffer for 30 min at 100 μl/well. Negative controls wereincubated with plain CSN instead of Mab-containing CSN and processed inparallel. Optical densities of 0.05 above the negative controls after 30min of substrate incubation time (SIT) were considered to be positiveresults:

As shown in FIGS. 1-4 and summarized in Table 2, all Mabs—except Mab9B4—were found to cross-react with spores and mycelia of multiple fungalspecies.

TABLE 2 Summary of species profiles of disclosed monoclonal antibodiesMab Positive reactivity to spores Positive reactivity to mycelium 1D4Fox, Mec, Mve, Sal, Sbi, Sch, Mec, Sch, Tha Scy, Sec, Smi, Sne, Ssu,Tha, Ska 3B2 Apu, Ccl, Che, Csp, Mec, Mve, Ccl, Mec, Mve, Sch Sal, Sbi,Sch, Scy, Sec, Smi, Sne, Ssu, Ska 4E12 Apu, Ccl, Che, Csp, Mec, Mve,Ccl, Mec, Sch Sal, Sbi, Sch, Scy, Sec, Smi, Sne, Ssu, Ska 9B4 Sch None9F9 Aca, Ani, Asy, Apu, Ccl, Che, Ccl, Mec, Sch Csp, Mec, Mve, Rst, Sal,Sbi, Sch, Scy, Sec, Smi, Sne, Ssu, Ska, Uch 10A5 Apu, Ccl, Che, Csp,Mec, Mve, Ccl, Mec, Mve, Sch Sal, Sbi, Sch, Scy, Sec, Smi, Sne, Ssu, SkaThus, Mab 9B4 can be used to species-specifically detect the presence ofStachybotrys chartarum spores in samples. The other five Mabs, whilereacting with all tested Stachybotrys and Memnoniella species, showcharacteristic reaction patterns with other fungi and can be classifiedinto 3 distinct groups. In group 1, Mabs 9F9 and 4E12 react with sporesand mycelium of all Cladosporium species tested, but react only withspores, and not mycelium, of Aureobasidium and Myrothecium. Both Mabsalso show some minor reactivity to spores of some other species. Ingroup 2, Mabs 3B2 and 10A5 show similar reactivity patterns whencompared to group 1, but in addition to spores, these Mabs alsorecognize mycelium of Myrothecium. This reaction pattern and the sporespecificity of Mab 9B4 indicate that fungal spores and mycelium expressunique epitopes, and that the above immunization protocol with sporescan be used as a method to generate spore-specific Mabs. Group 2 Mabsalso are more sensitive than group 1 Mabs. Since both Mabs in each groupshow very similar reactivity patterns to each other, and due to the factthat in each case they were derived from a single mouse, these Mabsmight well represent clones of the same plasma cell activated during theimmune response in each of these mice to Stachybotrys chartarum. Ingroup 3, Mab 1D4 reacts with spores and mycelium of all Stachybotrys andMemnoniella species and also reacts with spores and mycelium ofTrichoderma, but does not react with Cladosporium species. Mab 1D4 alsoconfirms antigenic differences between spores and mycelium by reactingwith spores, but not mycelium, of Myrothecium.

FIG. 2 not only shows that Mab 9B4 does not react with mycelium ofStachybotrys chartarum, but also shows that the reactivity of all otherMabs varies according to, different isolates of the fungus. Since theamount of antigen was standardized to 10 μg/ml for all twelve isolates,the differential Mab reactivity indicates that the antigenic compositionof individual isolates varies either due to absolute or relative numbersof antibody-reactive epitopes per unit biomass in the original mycelialextract.

FIG. 5 shows the sensitivity of Mab 9B4 against spores of Stachybotryschartarum in ELISA. It can be seen that the lower and upper values ofthe linear range of detection are 2000 and 40,000 spores/well,respectively. This degree of assay sensitivity is significantly betterthan that reported for Mabs produced against fungal allergens ofAlternaria alternata, in which case 10⁵ to 10⁶ spores were required forthe detection of the fungus. Vailes, L., et al., Journal of Allergy andClinical Immunology 107:641-646 (2001). However, it is less than thereported 5 to 400 germinating spores detectable by Mabs againstAlternaria brassicae, which were also produced against whole spores andspore germlings rather than individual molecules or allergens.Schmechel, D., et al., Food and Agricultural Immunology 9:219-32 (1997).These results indicate that whole spores, rather than isolatedmolecules, may be the antigen of choice to produce specific and highlysensitive Mabs against fungi.

FIG. 6 illustrates the effects of antigen boiling and periodateoxidization for 4 hours at RT with 20 mM sodium m-periodate in 0.1 Msodium acetate buffer, pH 4, on ELISA reactivity of all Mabs. It can beseen that boiling completely inhibits the reactivity of Mab 9B4 (IgG₁),while some binding activity remained after periodate treatment. Theseresults indicate that the epitope of Mab 9B4 is a protein or aglycoprotein. On the other hand, while boiling reduced the reactivity ofall other Mabs, periodate oxidation almost completely inhibited theirbinding. These results, and the fact that these Mabs are all of the IgMisotype, indicates that their epitopes are based on carbohydrates, notproteins.

Example 3 Purification of Mab 9B4

Mab 9B4 will be purified and concentrated from culture supernatants oftissue culture plates or flasks and from supernatants harvested from theextra capillary space of a CELLMAX artificial capillary cell culturesystem (Spectrum Laboratories, Rancho Dominguez, Calif.). For tissueculture plates, cells are allowed to reach confluence, and when 70-80%of the cells are dead, supernatant is harvested by centrifugation at1000 g for 5 min and passed sequentially through a 0.45 μm and a 0.22 μmfilter (Whatman, Clifton, N.J.), respectively. For the CELLMAX system,hybridomas are grown at cell densities of 1-10×10⁸ per cartridge andharvested periodically. Supernatants are combined, filtered as above,and used for Mab purification.

Mabs are purified using protein G and protein L (Sigma, St. Louis, Mo.)affinity chromatography. Protein G is used to deplete bovineimmunoglobulins from fetal calf serum prior to its use for tissueculture of hybridomas, thus producing Mab culture supernatants free ofbovine IgG. Darby, C. R., et al., Journal of Immunological Methods159:125-29 (1993). Protein L is used to purify and concentrate Mab 9B4according to the protocols of Harkins (Basic Methods in AntibodyProduction and Characterization, Howard, G. C., and Bethell, D. R.(Eds.), CRC Press, Boca Raton, Fla., pp. 141-168) and Heelan (Methods inMolecular Medicine 40:281-88 (2000)).

Example 4 Purification of Stachybotrys chartarum-Specific Antigen UsingMab 9B4

Specific antigen will be purified using Mab 9B4-mediated affinitychromatography. Mab 9B4 is covalently linked to Protein G beads (Sigma,St. Louis, Mo.) using a cross-linking, bi-functional coupling reagent,such as dimethyl pimelimidate, and established protocols. Harlow, E.,and Lane, D. (Eds.), 1998. The Mab 9B4-beads are then incubated with thesupernatant of sonicated spores of Stachybotrys chartarum, which willhave been previously incubated overnight on a rocking platform. Thebeads are packed into a column and antigen is collected by elution.

Example 5 Detection of Stachybotrys chartarum-Specific Antibodies inAnimal Samples

Stachybotrys-specific antibodies will be detected by direct ELISA or bycompetitive inhibition ELISA.

For direct ELISA, ELISA plates will be coated at 10 μg/ml of purifiedantigen and animal antibodies will be detected using the ELISA formatdescribed in Example 2, with titrated animal serum instead of Mab CSNand animal-specific biotin antibody conjugates (JacksonImmuno Research,West Grove, Pa.).

For inhibition ELISA, ELISA plates are also coated with 10 μg/ml ofpurified antigen and Mab 9B4 reactivity in competition with titratedanimal serum is analyzed using the ELISA described in Example 2. Thedegree of 9B4 inhibition is proportional to the amount of specificantibodies present in the animal serum. Since the antigen was purifiedwith the Stachybotrys chartarum-specific Mab 9B4, the antigen canlikewise be considered to be Stachybotrys chartarum-specific and, inturn, establish exposure to the fungus following a positive inhibitiontest.

Mimotopes, isolated from constrained phage display libraries (PhageDisplay: A Laboratory Manual, Barbas, C. F. (Ed.), Cold Spring HarborLaboratory Press, 2001; Phage Display of Peptides and Proteins, Kay, B.K., et al. (Eds.), Academic Press, San Diego, Calif., 1996) also will beexplored in the direct and inhibition ELISA to provide epitope-specifictests.

Having illustrated and described the principles of the invention byseveral embodiments, it should be apparent that those embodiments can bemodified in arrangement and detail without departing from the principlesof the invention. Thus, the invention as claimed includes all suchembodiments and variations thereof, and their equivalents, as comewithin the true spirit and scope of the claims stated below.

1. An isolated 9B4 monoclonal antibody wherein the antibody binds toStachybotrys chartarutn.
 2. A kit for detecting the presence ofStachybotrys chartarum, comprising a container containing the monoclonalantibody according to claim
 1. 3. The kit according to claim 2, furthercomprising instructional material.
 4. The monoclonal antibody of claim1, wherein the antibody is labeled.
 5. A hybridoma cell line thatsecretes the 9B4 monoclonal antibody as set forth in claim 1 depositedon Aug. 9, 2002 with the American Type Culture Collection underaccession number PTA-4582.
 6. A method of detecting Stachybotryschartarum, comprising: contacting the antibody of claim 1 with a sample;and detecting a complex comprising the antibody bound to an antigen fromStachybotrys chartarum.
 7. The method according to claim 6, wherein theantibody is immobilized.
 8. The method according to claim 6, whereindetecting further comprises contacting the antibody with a secondarylabeled antibody conjugate.
 9. The method according to claim 6, whereinthe sample is from an environment.
 10. The method according to claim 6,wherein the sample is obtained from an animal.
 11. The method accordingto claim 10, wherein the animal is a mammal.
 12. The method according toclaim 11, wherein the mammal is a human.
 13. The method according toclaim 11, wherein the sample is obtained from a lung.
 14. A method ofdetecting Stachybotrys chartarum, comprising: contacting the antibody ofclaim 4 with a sample; and detecting a complex comprising the antibodybound to an antigen from Stachybotrys chartarum.
 15. The methodaccording to claim 14, wherein the sample is from an environment. 16.The method according to claim 14, wherein the sample is obtained from ananimal.
 17. The method according to claim 14, wherein the animal is amammal.
 18. The method according to claim 17, wherein the mammal is ahuman.
 19. The method according to claim 15, wherein the sample isobtained from a lung.